Nucleotide selectivity is at the heart of the problem of understanding the effectiveness of nucleoside analogs used to treat HIV infections, and the evolution of resistance. In order to continue to develop new drugs and most efficiently use the ones available, it will be critical to understand the mechanisms by which HIV reverse transcriptase (RT) achieves nucleotide selectivity during DNA polymerization and how the enzyme changes in evolving to increase selectivity against nucleoside analogs while retaining sufficiently efficient incorporation of normal nucleotides. There has been considerable debate over the role of conformational changes in contributing to the selectivity of DNA polymerases as well as for other enzymes. New data suggest that a conformational switch dictates whether a dNTP will be incorporated or rejected. In this proposal we will investigate whether HIV RT follows this new paradigm for DNA polymerase selectivity. In preliminary data, we show that we can label HIV RT with a fluorophore on the fingers domain in a position that provides a signal to monitor the conformational changes upon nucleotide binding, and we present data to define the rates of nucleotide-induced changes in structure that precede incorporation. We will exploit this new signal to establish the pathway of reactions governing selectivity by HIV RT and define the role played by enzyme conformational changes in discrimination against nucleoside analogs. In addition, we will use this signal to examine changes in the dynamics of nucleotide binding and incorporation that underlie resistance to nucleoside analogs. We will also explore the mechanism by which nonnucleoside inhibitors alter dynamics of nucleotide binding and attenuate chemistry at the active site. These studies will be achieved using a combination of kinetic and structural methods, including stopped-flow fluorescence, rapid chemical quench-flow and single molecule fluorescence kinetic studies. This work will define better the reactions governing nucleotide selectivity by HIV RT, allow us to more rigorously interpret observed changes in enzyme structure thought to be responsible for resistance to nucleoside analogs, and provide insights to into the design and evaluation of new drugs needed to manage HIV infections. PUBLIC HEALTH RELEVANCE: Nucleotide selectivity is at the heart of the problem of understanding the effectiveness of nucleoside analogs used to treat HIV infections, and the evolution of resistance. This work will define the elementary steps governing nucleotide selectivity by HIV RT, allow us to more rigorously interpret observed changes in enzyme structure responsible for resistance to nucleoside analogs, and provide insights to into the design and evaluation of new drugs needed to manage HIV infections.